This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Little is known about the assembly of basal bodies and centrioles. We are using Chlamydomonas as a model system to identify and characterize the molecules important for basal body structure and function. Over the past year, we have used improved methods of specimen preparation and dual-axis electron tomography to study the structure and organization of basal bodies in the unicellular alga, Chlamydomonas reinhardtii. This work has revealed novel structures in wild-type and in strains with mutations affecting specific tubulin isoforms. Tomographic reconstructions of basal bodies from the delta-tubulin deletion mutant, uni3-1, have confirmed that basal bodies contain mostly doublet microtubules. Moreover, the stellate fibers that are present only in the transition zone of wild-type cells repeat within the core of uni3-1 basal bodies. The distal striated fiber is incomplete in this mutant, rootlet microtubules can be misplaced, and multiflagellate cells have been observed. A suppressor of uni3-1, designated tua2-6, contains a mutation in alpha-tubulin. In combination with the delta-tubulin deletion tua2-6;uni3-1 cells build both flagella, yet they retain defects in basal body structure and rootlet microtubule positioning. These data suggest that the presence of specific tubulin isoforms directly affects the assembly and function of basal bodies and associated structures in Chlamydomonas. This work has been published (O'Toole et al., Mol. Biol. Cell 14: 2999-3012, 2003). We are now pursuing analysis of strains that have mutations in the BALD2 gene and a new gene, TNS1 that is important in basal body assembly. The Dutcher lab has recently found that the BALD2 gene encodes epsilon-tubulin. To further understand the role of BALD2 in basal body morphogenesis, we are analyzing strains with new bald2 alleles as well as extragenic suppressors that partially restore basal body structure and function.